Anticancer activity, phytochemical investigation and molecular docking insights of Citrullus colocynthis (L.) fruits

Cancer disease is regarded as one of the most significant public health issues, regardless of economic standards. Medicinal plants are now regarded as a natural source of anticancer medicines due to their antioxidant and anti-mutagenic actions. Cucurbitaceae is considered to be one of the most economically significant families. One family species is Citrullus colocynthis (L.), which has a high concentration of many active secondary chemical metabolites. Various C. colocynthis plant extracts showed cytotoxicity against some cancer cells. This study aims to identify the C. colocynthis fruit components and determine whether they have anticancer action against MIA PaCa-2 and A431 cells. High-Performance Liquid Chromatography/Quadrupole Time of Flight/Mass Spectrometry (HPLC/QTOF/MS); the technique was accustomed to investigate the compounds of the ethyl acetate (EtOAc) fruit extract. Anticancer activity was investigated on both MIAPaCa-2 and A-431 cell lines. DPPH assay for antioxidant activity was carried out. Molecular modelling was employed to help understand the molecular basis for the observed anticancer activity. 24 compounds were tentatively identified by comparing the extract’s fragmentation pattern in positive mode against reference compounds spectra and literature. The EtOAc extract of C. colocynthis had effective positive results on cancer cells (MIAPaCa-2 and A-431) and was characterized by slight or no harmful effect on normal (healthy) cells. For the DPPH assay, EtOAc and BuOH extracts exhibited high antioxidant activity (86 and 76%, respectively) compared with the oxidative potential of the standard compound (Caffeic acid, 98%). One of the major cucurbitacin derivatives that LC/MS tentatively identified in the EtOAc extract was Cucurbita-5(10),6,23-triene-3β,25-diol. During this study, docking experiments and MD simulations were carried out, which suggested the anti-pancreatic cancer activity of C. colocynthis extract to be attributed to EGFR inhibition by Cucurbita-5(10),6,23-triene-3β,25-diol. Therefore, expansion of this type of research should be encouraged in the hope of obtaining natural therapeutics for cancerous tumors in the future, having the advantage of being cheaper, safer, and with fewer side effects.


Materials and methods
In this study, all methods followed the relevant guidelines/ regulations/ legislation.

Preparation of plant extract
Around June 2022, the C. colocynthis (L.) fruits were purchased from a local Egyptian market.After the plant material was dried in an oven at 40 °C for five days, it was ground into powder using an electric mill.Until needed, the powdered sample was stored in an airtight container.A soxhlet extracted about 450 g of the powdered dry fruits over 72 h using solvents (2.5 L each) with increasing polarity.The plant extracts were concentrated in a rotary evaporator at 40-60 °C, and the finished extracts were stored in the fridge.Hexane (3 g), dichloromethane (8 g), ethyl acetate (13 g), butanol (11 g), and aqueous layer (21 g) were the five fractions that were obtained.

HPLC-ESI-QTOF-MS/MS
HPLC-ESI-QTOF-MS/MS technique is used to look into the secondary metabolites of C. colocynthis fruit ethyl acetate extract.50 mg of extract was dissolved in 1 mL of mobile phase A (water 0.1% formic acid), vortexed for 2 min, ultrasonically disrupted for 10 min, and centrifuged for 5 min at 10,000 rpm.The gas temperature and drying gas flow were 200 OC and 8 l/min, respectively.The injection had a 3 g/l concentration.The multi-step linear gradient was applied to mobile phase A (water 0.1%formic acid) at a flow rate of 3 ml/min for 45 min, starting at (90-10) percent of mobile phase B (Acetonitrile 0.1%formic acid).Columns: Agilent Technologies pre-Zorbax RP-18 column (dimensions: 150 mm 3 mm, dp = 2.7 m).For separation, a column at a temperature of 200 °C was utilized.The instrument used for chromatographic separation at the Faculty of Pharmacy-Fayoum University was the 6530 Q-TOF LC/MS (Agilent Technologies) equipped with an autosampler (G7129A), a quaternary pump (G7104C), and a column comp (G7116A).With a capillary voltage of 4500 V, ESI's ( +) ionization mode was used to acquire mass spectra.The mass spectra were captured between 50 and 3000 m/z.The collision energy was set to 10 V, while the skimmer and fragmentation voltages were set to 65 and 130 V, respectively.

Cytotoxicity
Cell culture.Human pancreatic cancer cell lines (MIAPaCa-2) and human skin cancer cells (A-431) were obtained from the American Type Culture Collection (ATCC).The cells were then cultured in T-25 flasks (Jet Biofil Flask) using 4-6 mL of DMEM-high glucose medium containing 10% fetal bovine serum (Gibson, 26140-079) and 1% antibiotic of penicillin-streptomycin (Gibson, 15140-122).They were raised at 37 °C in a 5% CO 2 environment.The subculture's cells were washed twice with 1 ml PBS after removing the covering media when they were 80% confluence.The cells were pre-incubated for 1-2 min with trypsin-EDTA 0.25% (Gibson, 25200-056), and then the trypsin effect was countered by adding 3 ml of DMEM medium.They were put into 15 ml Falcon tubes and centrifuged at 1700 rpm for 7 min.The cells were pelleted, the supernatant medium was removed, the fresh medium was added, and the cells were placed in incubation flasks. 1 ml of freezing media (5-10% DMSO with 90-95% FBS) was used for freezing.
Using the MTT assay, determine cell toxicity.The colorimetric [3-(4, 5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazolium bromide] tetrazolium reduction assay (MTT) was used to assess the vitality of the cells.A 96-well plate was used to seed the MIAPaCa-2 and A-431 cell lines, and 48 h of incubation were required.After incubation, the cells were treated to a final concentration of 100 ppm of the tested medicines in triplicates.180 µl of the new medium was introduced to each well after the existing medium had been removed after 48 h.The MTT assay described by Mosmann 15 evaluated cytotoxicity.After treatment, the medium is removed from each well, and 20 µl of MTT (Sigma, Germany) is added.Each well is then incubated for 4 h at 37 °C in the dark.Following incubation, 200 µl of DMSO was added in place of the MTT solution.The cells were then placed in a shaking incubator for 10 min.An ELISA plate reader measured absorbance at 570 nm after adding glycine buffer.
Determination of IC 50 values.Several concentrations of highly active samples that exhibited 60% cytotoxicity on various cancer cell lines were generated for dose-response investigations.The findings were utilized to determine the IC 50 values of each sample in comparison with doxorubicin, a positive control.

DPPH assay
In the wells of a 96-well plate, 20 µl of extract that had been suitably diluted in DMSO was combined with 180 µl of DPPH in methanol (4 mg/ml) for the DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging experiment.After 15 min of darkness, the plate was placed in a Multiskan automatic kinetic microplate reader (Labsystems Multiskan RC reader) to measure the solution's absorbance at 540 nm.Standard (Trolox solutions in DMSO) and appropriate blanks (DMSO) were run simultaneously.The EC 50 (the concentration that reduces DPPH absorbance by 50%) was determined by testing the extracts at different concentrations.This approach closely resembles that of earlier researchers 16 .Testing was done in triplicate.

Molecular docking
Based on the literature, EGFR was selected as a drug target for pancreatic cancer 17 .The X-ray crystal structure of EGFR and its co-crystallized ligand (PDB ID: 5X2A) 18 was obtained from the Protein Data Bank.The protein and the 3D structure of Cucurbita-5 (10),6,23-triene-3β,25-diol were prepared using the structure preparation wizard in MOE (version 2019.01) 19, and docking was performed using GOLD (version 5.8) 20,21 using the ChemPLP scoring function as previously described 22,23 .PyMol was used for the generation of all figures 24 .

Molecular dynamics
A 100 ns MD simulations was carried out using the PMEMD.cudacode of the AMBER Molecular Dynamics package 25 for EGFR-Cucurbita-5(10),6,23-triene-3β,25-diol complex following the same previously described protocol of minimization, heating, density equilibration and production 26 .The trajectories were analyzed using CPPTraj 27 .Plots and visual inspection of the trajectories were done using XMgrace 28 and VMD 29 , respectively.

Ethics approval and consent to participate
The study was commenced after ethical clearance was secured from the National Research Centre Ethical Committee, Egypt, with protocol number (6441112202).

Viability assay by MTT
The viability of the MIAPaCa-2 and A-431 cell lines was evaluated using the MTT test after treatment with 100 µg/ml of C. colocynthis extracts.As seen in Fig. 1, the EtOAc fraction showed the highest cytotoxic effect, inhibiting PaCa-2 and A-431 by 54.4 and 68.3%, respectively.On the other hand, the extract showed only 1.3% inhibition on normal cells (BJ-1), indicating the extract to be selectively cytotoxic to cancer cells and not normal cells.
Additionally, the EtOAc extract demonstrated an IC 50 value of 17.4 0.12 and 13.1 0.21 µg/ml of PaCa-2 and A-431 cancer cells, respectively.The cytotoxicity is nearly 2.3 and 1.3 folds more potent than the IC 50 value obtained with the positive control drug, doxorubicin (21 1.2 and 37.6 1.5 µg/ml, respectively) (Table 1).

DPPH test
Comparing the oxidative potential of the reference chemical (Caffeic acid, 98%) utilized in this study to the antioxidant activity of the EtOAc and BuOH extracts revealed their strong antioxidant activity in the DPPH experiment.The antioxidative activity of the EtOAc and BuOH extracts was significantly high (86% and 76%, respectively) with relative EC 50 values of 1.0 and 0.62 mg/ml, respectively (Fig. 2).

HPLC/ESI/MS/MS annotations of the main compounds of C.colocynthis ethyl acetate fruit extract
In the current study, the chemical composition of the EtOAc extract was analyzed utilizing the positive ionization mode of the LC-ESI-QTOF-MS/MS method.By comparing the fragmentation pattern in the positive mode (Fig. 3) against the spctra of the reference compounds and literature, a total of 24 molecules shown in Table 2 were tentatively identified.The extraction solvent greatly impacts the chemistry of the materials under study.The limit of detection for each peak of chemicals was calculated using the Rt and whole MS spectra.

Molecular docking
EGFR was previously reported to be over-expressed in pancreatic cancer, representing an attractive therapeutic target 17 .Accordingly, we attempted to check the binding mode of the major compound of C. colocynthis extract to EGFR.The structure of EGFR complexed with the N8-phenyl-9H-purine-2,8-diamine reversible inhibitor, SKLB(3) (PDB code: 5X2A) 18 , was used to check the binding mode of Cucurbita-5 (10) check the reliability of the resultant docked poses of the docking protocol, the co-crystallized ligand, SKLB (3), was docked into the active site of EGFR.Overlay of the docked pose and the experimentally determined position showed a similar binding mode with a root mean square deviation (RMSD) value of 0.78 Å (Fig. 4A).SKLB (3) occupies the ATP-binding cleft of EGFR, which is mainly lined by hydrophobic residues, with both ends of the aperture lined by polar residues.SKLB (3) completely occupied the aperture, forming several hydrophobic interactions and a few direct and water-mediated H-bonds.Substituents on the N-9 position of purine are sandwiched between Leu718, Val726, and Leu844 sidechains (Fig. 4A).These three residues form what has been previously reported as a "hydrophobic clamp," playing a crucial role in the EGFR binding of reversible inhibitors 18 .
The binding orientation of Cucurbita-5(10),6,23-triene-3β,25-diol is similar to SKLB (3).Its hydrophobic cyclopentaphenanthrene ring, representing the core scaffold of the compound, is sandwiched between the three "hydrophobic clamp" residues (Fig. 4B).The twist in the binding pose of the cyclopentaphenanthrene scaffold makes the cyclopentyl ring mimic the N-9 substituent of SKLB (3), which is believed to be crucial for EGFR tight binding 18 .Additionally, it is anchored from each end with a H-bond interaction between (1) the hydroxyl group of the heptenyl chain and the backbone carbonyl of Pro794 from one end and (2) the 3-hydroxyl group of the cyclopentaphenanthrene ring and the sidechain of Met790 from another end.
To confirm the stable binding of Cucurbita-5 (10),6,23-triene-3β,25-diol to EGFR, a 100-ns MD simulation for the complex was carried out using AMBER software package 25 .The resultant trajectory was visually examined which showed the retention of Cucurbita-5 (10),6,23-triene-3β,25-diol within the bidning site of EGFR (Fig. 5A).The initial docking binding mode was confirmed by RMSD analysis of the ligand nonhydrogen atoms which showed little deviation from the initial geometry with a plateau observed at 2 Å after 40 ns (Fig. 5B).Similarly, EGFR maintained its conformation with little deviations observed in the RMSD of

Discussion
Cancer is regarded as one of the most significant public health issues, regardless of the economic standards 1,2 .Medicinal plants are now regarded as a natural source of anticancer medicines due to their antioxidant and antimutagenic activities along their low side effects, low cost, and ease of availability.Accordingly, medicinal plants are viewed as an attaractive option for cancer treatment 4 .
This study focused on evaluating the cytotoxic effect of C.colocynthis on pancreatic and skin cancer cells.The EtOAc extract of C. colocynthis demonstrated significantly favorable effects on cancer cells (PaCa-2 and A-431) while having little to no negative effects on healthy (normal) cells.This cytotoxic selectivity represents a major advantage for the EtOAc extract of C. colocynthis over other chemotherapeutic agents that lack this selectivity and kill both cancer and normal cells.Our results appear in agreement with previous studies that showed C. colocynthis plant extracts to have cytotoxic effects against some cancer cells 1,2 .Perven et al. 12 reported that the ethanolic fruit extract had cytotoxic effects on the HEp-2 and L929 cancer cell lines in Wistar mice.Alkaloids from fruit extracts had cytotoxic effects against human breast cancer cell lines (MCF-7, HepG-2) 11 .The leaf extracts showed cytotoxic effects and induced apoptosis on the ER-MDAMB-231 and MCF7 cell lines which were used to model human breast cancer 8 .
Chemical analysis of the EtOAc extract of C. colocynthis was conducted by comparing the fragmentation patterns in the positive mode to reference compounds' spectra available in literature.Accordingly, a total of 24 compounds were identified with Cucurbita-5(10),6,23-triene-3,25-diol, a derivative of cucurbitacin (a highly oxygenated tetracyclic triterpene) identified as a major component in the extract.Molecular docking experiments showed Cucurbita-5(10), 6,23-triene-3,25-diol to bind effectively in the binding site of EGFR with a 100-ns MD simulation further proving the stability of this complex.The modelling studies strongly suggest Cucurbita-5(10), 6,23-triene-3,25-diol binding to EGFR to be responsible for the observed anticancer activity of the EtOAc extract of C. colocynthis.Our results align with previous studies reporting the role of Cucurbita-5(10),6,23-triene-3,25-diol in cancer treatment 34,35 .
In conclusion, the results show a selective cytotoxic effect of the the EtOAc extract of C. colocynthis towards pancreatic and skin cancer with Cucurbita-5(10), 6,23-triene-3,25-diol being a major component contributing to this observed effect.This suggests C. colocynthis to be a cheap and safe therapeutic to be used in pancreatic and skin cancers.

Figure 2 .
Figure 2. Concentration-response curves for the DPPH radical scavenging activity of Caffeic acid (positive control), different fractions of C.colocynthis.

Figure 3 .
Figure 3. HPLC/MS/MS base peak chromatogram of C.colocynthis fruits in positive ionization mode.